Homogeneous Electrochemical Strategy for Human Telomerase Activity Assay at Single-Cell Level Based on T7 Exonuclease-Aided Target Recycling Amplification

As an important biomarker for early cancer diagnostics and a valuable therapeutic target, telomerase has attracted extensive attention concerning its detection and monitoring. Herein, a homogeneous electrochemical strategy based on T7 exonuclease-aided target recycling amplification is proposed for a simple, rapid, and highly sensitive assay of human telomerase activity from crude cancer cell extracts. In this strategy, a 5' methylene blue (MB)-labeled hairpin (HP) probe is designed, which can hybridize with the telomerase reaction products to initiate the subsequent digestion by T7 exonuclease, and a large amount of MB-labeled mononucleotides are released to result in the significantly amplified electrochemical signal. By taking advantage of the high amplification efficiency of T7-aided target recycling, the present assay enables the detection of telomerase activity at the single-cell level, which is superior or comparable to that of the reported literature. Furthermore, the assay was carried out in a homogeneous solution without complex modification or immobilization procedures, which has the merits of simplicity, rapid response, and improved recognition efficiency compared with heterogeneous biosensors. With the ability of fast detection, outstanding sensitivity, and excellent selectivity, this strategy offers a convenient and specific method for telomerase activity detection, which exhibits great potential in the practical application in telomerase-based early stage cancer diagnosis.